Search

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol PEth—a biomarker for ethanol uptake—has been developed by LCESIMSMS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol PBut as internal standard. PEth homologues with two fatty acid substituents—PEth 18 : 118 : 1, PEth 16 : 016 : 0—were determined in postmortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 gdose of ethanol. Furthermore, PEth 18 : 116 : 0 or its isobaric isomer PEth—16 : 018 : 1 was detected. In comparison to previous highperformance liquid chromatography HPLC methods with evaporative light scattering detection ELSD, the LCMSMSmethod is more sensitive—with a limit of detection below 20 ngml—and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LCMSMS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLCELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LCMSMS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLCELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate. Copyright © 2009 John Wiley & Sons, Ltd.

Abstract: A striking difference was observed for cellular-bound drug in HaCaT and Sk-Mel-1 cells for a fixed drug exposure time of 72 h and varying 3H-haloperidol concentrations in the culture media. Drug uptake was dependent on drug concentration and linearly correlated for both the non-pigment- and the pigment-producing cells which however was different in magnitude. In an additional investigation the time course of drug uptake during 3H-haloperidol exposure (400 pmol/ml; 28 days) revealed increasing drug concentrations in the Sk-Mel-1 population, whereas drug concentrations in the keratinocytes reached a plateau within a short time period. In contrast to the HaCaT cells no tendency to saturation was observed for the pigment-producing cell line. At the end of the experiments 3H-haloperidol concentrations in Sk-Mel-1 were found to be approximately tenfold higher than in HaCaT.

Abstract: In view of the melanin-binding characteristics of haloperidol and its differential uptake by pigment- and non-pigment-producing cells, a co-culture of HaCaT with Sk-Mel-1 cell lines was performed to investigate whether melanosomes act as carriers for drug molecules associated with the pigments. Initially, HaCaT and Sk-Mel-1 cells were separately cultivated in the presence of 3H-haloperidol (400 pmol/ml medium ) for 28 days followed by subsequent co-cultivation in the absence of 3H-haloperidol for 5 days. The transfer of pigments into the keratinocytes during co-culture was confirmed by transmission electron microscopy. After the co-culture experiments a striking increase (≥ 50%) of 3H-haloperidol was observed in the pigmented HaCaT cells compared to the unpigmented keratinocytes. The present study proved the role of pigments as carriers for melanin-associated drug molecules. The results supported the hypothesis that hair pigment might be a factor affecting the outcome of hair assays for particular categories of commonly used licit and illicit substances. The chosen cell lines and the developed co-culture system may represent suitable in vitro models to study differential drug uptake into cell populations present in the skin or in the growing hair follicle as well as to elucidate drug uptake due to melanocyte-keratinocyte interactions.

Abstract: In the present study, concentrations of dihydrocodeine and its metabolites in saliva and serum were compared after single low-dose and chronic high-dosage administration of the drug. In the first investigation, blood and saliva were collected periodically from six subjects after oral administration of 60 mg dihydrocodeine. In the second study, 20 subjects on oral dihydrocodeine maintenance provided single samples of blood and saliva simultaneously. Serum protein binding of salivary analytes and their recovery from the adsorbing material of the collection device as well as pH values of saliva samples were determined. The fluids were analyzed for dihydrocodeine and the major metabolites by high-performance liquid chromatography. In the single dose study dihydrocodeine was the only analyte found in saliva for up to 12–24 h post-dose. The half-life of dihydrocodeine in saliva was about twice that found in blood. The ratios of saliva/ serum concentrations ranged from 1.2 to 17.0. After chronic high-dosage use, dihydrocodeine was the main salivary analyte and N-nordihydrocodeine was present in a few samples. Saliva/serum concentration ratios of dihydrocodeine were strongly dependent on the pH value of saliva and, to a lesser extent, on serum-protein binding. The saliva/ serum ratios were more similar after chronic administration. The data suggest a passive diffusion process as the underlying mechanism for the transport of dihydrocodeine into saliva. After both single and chronic use, the presence of the drug in saliva can be used as evidence of recent substance administration.

Abstract: The influence of the special shampoo Ultra Clean (Zydot Unlimited, Tulsa, Oklahoma) on the results of hair analyses was investigated. Hair samples from persons (n = 14) with a known history of drug abuse were collected at autopsy. The hair samples were divided into separate strands which were analyzed both after washing with Ultra Clean and without treatment. Hair analyses were performed by methanol extraction under sonication, purification by solid phase extraction and GC/MS in SIM mode according to routine procedures for tetrahydrocannabinol (THC), cocaine, amphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDE), heroin, 6-monoacetylmorphine (6-MAM), morphine, codeine, dihydrocodeine and methadone. All drugs originally present in the hair fibers were still detected after a single ¶application of Ultra Clean. However, a slight decrease in drug concentrations could mostly be observed e.g. cocaine (n = 10) –5%, 6-MAM (n = 12) –9%, morphine (n = 12) –26%, THC (n = 4) –36%. The findings clearly demonstrated that drug substances had not been sufficiently removed from human hair by a single Ultra Clean treatment to drop their concentrations below the limit of detection of the analytical method applied.

Abstract: Various aspects concerning the practical application and forensic interpretation of data obtained by saliva drug testing and drug monitoring from the skin surface are discussed. Basic information on the composition of saliva and skin secretions and their particular transport mechanisms, as far as known, are given. For drugs of abuse secretion into saliva is suggested to be by passive diffusion and to depend on lipid solubility, pKa, plasma protein binding and on the pH of saliva. Drug molecules from blood are considered to reach the skin surface by various routes such as by sweat and sebum as well as by inter- and/or transcellular diffusion. The role of the stratum corneum as a temporary drug reservoir exceeding positive drug findings in urine is outlined. Current data on opioids, cocaine metabolites, cannabinoids and amphetamines detected in saliva and on the skin surface are reviewed. Aspects of collection, processing and analysis of the samples for implementation in roadside testing are addressed. The requirement of test sensitivity covering the broad concentration ranges and the importance of test specificity bearing in mind that the parent drug is the main analyte present in those specimens is stressed. Theoretical and practical findings on frequently abused drugs are discussed with regard to the possibilities and limitations of drug monitoring from saliva and perspiration to support a suspicion of actual or recent drug administration.

Abstract: The levels of dihydrocodeine found in impaired individuals and in fatalities show a wide overlap in the ranges. Among other factors, the genetically controlled metabolism of dihydrocodeine should play an important role in dihydrocodeine toxicity. For the first time, the most important metabolites of dihydrocodeine were investigated in femoral blood from three fatal cases by simultaneous determination using HPLC and native fluorescence for detection. The amount of parent drug always exceeded dihydrocodeine-glucuronide formation and dihydromorphine concentrations ranged from 0.16–0.21 mg/L. The similar binding affinities of dihydromorphine and morphine to μ-opioid receptors suggest similar pharmacological effects and adverse reactions. The determination of the pharmacologically active metabolites should help to clarify the cause of death in fatal cases especially if a relatively low concentration of the parent drug is found.

The postmortem distribution of morphine and its metabolites was investigated in four cases of heroin overdose to evaluate some of the factors that influence intravasal blood concentrations. Variables included were the chemical stability of morphine conjugates, hemoconcentration, incomplete distribution of the drug and diffusion processes. Blood samples from different sampling sites including the aorta, the infra- and suprarenal portion of the inferior vena cava, the superior vena cava, the femoral and subclavian veins, and the right and left ventricles were examined for morphine, morphine-3-glucuronide and morphine-6-glucuronide, hematocrit and water content. Drug concentrations were determined by HPLC based on the native fluorescence of the analytes. Morphine glucuronides proved to be stable for a time period of 72 h. The water content ranged from 65 to 83% and hematocrit values from 25 to 75%, and were seen as contributory factors to the dramatic differences observed for drug concentrations from different sampling sites. The differences could neither be attributed to incomplete distribution during life-time nor to a diffusion process following the different distribution volumes of morphine and its conjugates. A definite relationship between the ratio of the molar concentrations of morphine and its glucuronides, as assessed in pharmacokinetical studies after morphine dosing, could not be established. For a better understanding more cases and changes over time and tissue concentrations should be analysed.

Hair samples were taken at autopsy from the head of 1 male and 1 female subject both known as drug abusers. Some of the strands were bleached by in-vitro cosmetic treatment. The bleached hair as well as the original hair samples were partly exposed to water or soil prior to further investigations and drug monitoring. The exposure times were 4 weeks or 6 months for water and 6 months for soil. The hair fibers were examined by transmission electron microscope (TEM) and by scanning electron microscope (SEM) investigations. The electron microscope studies confirmed that all experimental conditions had produced morphological alterations in the hair fibers. After exposure to water or to soil for 6 months as well as after storage of the clipped bleached hair in tap water at room temperature for 4 weeks, drug monitoring of formerly positive hair samples gave negative results. After storage of natural hair in soil or in water for 4 weeks the opiate levels had dramatically decreased. The samples were screened by fluorescence polarization immunoassay after enzymatic digestion. The results were confirmed by GC/MS.

Abstract: An N2-laser of 19 ns pulse-duration and an energy of 30 mJ has been developed and examined. A dc preionization, a grooved electrode and a partial electrical screening of the discharge tube permit reproducible single-shot operation and yield a homogeneous beam.

The results of recent investigations of the analgesic and the nonanalgesic effects of opioid glucuronides are relevant to the research on drug abuse in forensic toxicology. As has been shown for heroin, knowledge of the state of distribution and elimination of active and inactive metabolites and glucuronides offers new possibilities in forensic interpretation of analytic results. Because of similar metabolic degradation, calculation of the time-dependent ratio of the concentration of morphine and its glucuronide metabolites in blood or serum allows a rough estimation of increased dosage and of time elapsed since the last application. Drug effects can be examined with respect to individual case histories, including overdose and survival time if the patient died. However, different methods of administration and the strong influence of different volumes or compartments of distribution of parent compounds and metabolites on concentrations in human body tissues require careful use of glucuronide concentration data.

Abstract: The in vitro experiments on the interaction of 3H-cocaine and melanin from Sepia officinalis confirmed the existence of drug binding sites on melanin granules. The results suggested that the binding of 3H-cocaine to melanin could be analyzed by assuming that the binding to the surface of pigment granules is analogous to the adsorption of a drug on a solid and follows Langmuir adsorption isotherm type I. Scatchard analysis indicated heterogeneity of binding sites. Structural and chemical alterations caused by isolation of the melanoproteins, which are heterogenous in nature and show different physicochemical properties, are considered to be most crucial. The studies on hair samples confirmed that melanin-drug interactions occur on the surface of melanin granules. These seem to be of minor importance compared to the drug-melanoprotein loading during melanogenesis for the observed influence of pigmentation on the drug content of hair fibers. From the results it was concluded that in vitro studies on melanin provide limited information and even drug-soaked hair must be regarded as inappropriate for the study of melanin-drug-binding in hair.

Abstract: An approach to determine the stability of benzodiazepines and some of their metabolites (n = 13) by means of a routinely applied gas chromatographic method using electron capture detection was made in this preliminary study. Validation data of the method are given. Spiked blood and plasma samples were stored at 4° C and analysed at selected times up to 240 days. The concentrations of all analytes had decreased to at least 60% of the original levels at the end of the observation period. A clear pattern of breakdown could not be established. The data obtained suggest that results from long-term stored samples should be interpreted cautiously. Further investigations concerning the stability of drugs in blood and plasma samples, additional methods of identification and determination as well as the establishment of optimal storage conditions seem necessary.

A venous blood sample taken at autopsy cannot be considered to represent the antemortem blood concentration of a particular substance. Autolytic processes cause disintegration and increasing permeability of the physiological and anatomical barriers such as vascular walls and lead to changes in substance concentrations. In the present study, the experimental design represents an in vitro postmortem simulation of a drug substance crossing a venous wall. The postmortem behavior of morphine, morphine-3- and morphine-6-glucuronide was investigated. A Chien-Valia-diffusion chamber with a patch of inferior vena cava as diffusion barrier was used. For optimal simulation of postmortem events, vein sampling was restricted to selected autopsy cases. Parameters for the analysis of diffusion across the vascular tissue were dependence on time, temperature, and initial substance concentrations. The penetration behavior simulating venous efflux and influx of the substances was studied by different orientation of the venous wall in the experiments. Rhodamine B was used as a model substance to visualize the binding to the tissue and the passage across the venous wall. The permeation of morphine, morphine-3- and morphine-6-glucuronide across a vein tissue was found to be mainly dependent on the disintegration of the vascular wall and on the postmortem time period as well as on concentration gradients. From the data of this preliminary in vitro study, it can be concluded that a lag time for transvascular diffusion exists postmortem. However, it could be demonstrated, that adsorption to and penetration into the vascular tissue may alter intraluminal blood concentrations even at an early stage of the postmortem time period.

A noninvasive and nonocclusive skin patch (Sudormed™) was investigated for the systematic collection of drugs of abuse over a period of several days. First, the applicability and user friendliness were tested by volunteers. The permeability of the polyurethane dressing from the outside to the inside for an aqueous solution was shown by incubating the outside layer with Rhodamine B. No fluorescence could be detected in the cotton pad beneath. A single dose experiment using theophylline as a model compound showed that there was a delay in time before the substance could be determined in the pad. The drug content decreased with increasing time of patch application. When eight volunteers participating in a methadone treatment were monitored, the substitute drug could always be detected in the patch associated with a minor concentration of EDDP. Besides, in some of the patches investigated, indications for an abuse of cocaine and heroin were found. The so-called sweat patch appears to be a valuable tool in clinical and forensic toxicology, as it offers a longer and prospective surveillance period compared with blood and urine testing.

Diffusion experiments were performed using clipped hair fibers as diffusion bridges and aqueous solutions of morphine, codeine and dihydrocodeine. Natural as well as predamaged hair fibers were investigated. The test series were conducted at ambient temperature and at high humidity. After 312 or 372 hours the middle segments of the strands were clipped, washed and analyzed by GC/MS. Only when virgin hair samples were used the solutes passed along the fiber at full length resulting in a positive immunological finding at the end of the diffusion bridge. Most of the washing fluids were positive for opiates. All centerpieces had a high opiate content. The opiate concentration in damaged hair was significantly higher. Radial swelling of the hair fiber with radial diffusion was the first and main process to appear when hair was exposed to water. The diffusion process in hair could not be placed in a simple mathematical treatment.

Tortoise shell guinea pigs (n= 7) were administered codeine (1 mg/mL codeine-base) in their drinking water for 3 weeks. Black, reddish-brown and white hair was collected separately from each animal before and after treatment. The hair samples were analyzed by GC/MS. The experiment showed positive results for all hair fibers with large individual variability of drug incorporation. Low drug intake resulted in small differences of the drug content in hair fibers different in color, whereas in cases of high drug intake a strong influence of hair pigmentation on the analytical results was observed. The highest drug content was always found in black hair samples, non-pigmented hair showed the lowest drug concentrations and the drug content in reddish-brown fibers was less than in black hair samples from the same animal. From the results it was concluded, that eumelanins rather than pheomelanins are the decisive factor for codeine-melanin binding in hair and the amount of drug intake was suggested to determine the relevance of hair pigmentation on the analytical results.

The kinetic profile of ethanol and ethyl glucuronide (EtG) in serum was investigated in three subject groups: 1) Healthy, moderately drinking volunteers (daily intake less than 30 g ethanol) who ingested a single dose of ethanol. In this group the maximum of serum ethyl glucuronide concentration (SEtGC) and of serum ethanol concentration (SEC) did not exceed 3.7 mg/L and 1.5 g/L respectively. EtG peaked 2 to 3.5 h later than ethanol. EtG was eliminated with a terminal half-life of 2 to 3 h. EtG decreased slower than ethanol—the metabolite could still be determined in serum up to 8 h after complete ethanol elimination. 2) In serum samples of teetotalers neither ethanol nor EtG could be found. 3) In 37 of 50 serum samples of drivers suspected of driving under the influence of ethanol, SEtGC was found between the limit of detection (0.1 mg/L) and 20 mg/L. If the SEC is less than 1 g/L and the SEtGC is significantly higher than 5 mg/L, we assume alcohol misuse.